The present invention relates to genes coding for eicosapentaenoic acid (designated EPA hereinafter) synthesizing enzymes, plasmids containing these genes, microorganisms transformed with the plasmid, and a process for production of eicosapentaenoic acid using the microorganism. The EPA is useful as a starting material for pharmaceuticals, foods, feeds and the like.

Polyunsaturated fatty acids represented by eicosapentaenoic acid (EPA) play an important roll as a component of a biomembrane. So far, the following pharmacological actions of EPA are known. (1) Platelet coagulation inhibitory action (thrombolytic action), (2) blood neutral fat-lowering action, (3) actions for lowering blood VLDL-cholesterol and LDL-cholesterol and increasing HDL-cholesterol (anti-arterial sclerosis action), (4) blood viscosity-lowering action, (5) blood pressure lowering action, (6) anti-inflammatory action, (7) anti-tumor action.

In addition, EPA is a substrate for biosynthesis of prostaglandins and exhibits an essential function in vivo in higher mammals including humans. In particular EPA is important as a substrate for production of three types of prostaglandins, has platelet coagulation inhibitory action and is studied for applications to treatment and prophylactic agents for thrombosis. In addition EPA has especially high activity for lowering plasma cholesterol level among polyunsaturated fatty acids having said action, and is highly effective in comparison with linoleic acid and the like usually contained in plant oil. Additionally, EPA is known as an essential nutrient for fish.

Thus, epidemiological research by Dyerberg, Denmark (Am. J. Clin. Nutur, 28, 959, 1975) showed a possibility for use as health foods or pharmaceuticals on the basis of thrombosis inhibitory action or lipid-lowering actions of EPA. However, as can be seen from its chemical structure, the chemical synthesis of EPA is very difficult. Accordingly, in Japan, it is recommended to eat blue back fish such as sardine, salmon, saury, and the like.

At present, most commercially available EPA products are fractionation products from fish oil obtained by a boiling process, and the EPA content thereof is about 10 to 30%. Fish oil extracted by a boiling process is a mixed glyceride containing various kind of fatty acids as component fatty acids, and not only is isolation and purification of each component difficult, but also since EPA is a polyunsaturated fatty acid having 20 carbon atoms and five double bonds all of which are cis-type, EPA is also a unstable and highly oxidation-liable fatty acid. Therefore, it is necessary for EPA to be concentrated from fish oil with prevention of oxygen, light, heat and the like. In addition, since fish oil contains various fatty acids in addition to EPA, their fractionation is difficult. Moreover, although various organic solvents used for fractionation of EPA are eliminated under a reduced pressure, complete elimination of the organic solvents is difficult from a technical and economical point of view.

Most EPA preparations used for pharmaceuticals are those having at least 90% EPA concentration, produced by extracting fish oil by various Processes, hydrolysing the fish oil enzymatically or under an alkaline condition to generate free fatty acids, optionally converting the free fatty acids to corresponding methyl or ethyl esters, and further purifying by fractional crystallization at a low temperature, applying a urea-addition method, distillation under a reduced pressure, reverse phase chromatography, or the like. However, since these processes use many organic solvents and heating to near 200°C, it is possible that an EPA concentrate obtained by using such process may be denaturated by residual organic solvent, and polymerization, isomerization or oxidation of EPA. Moreover, where a fish oil is used as a starting material for production of EPA, it is difficult to eliminate docosanoic acid or the like which is considered to be a cause of cardiodiseases, and therefore problems remain in the use for health foods, pharmaceuticals or the like.

On the other hand, recently, processes for production of EPA using microorganisms such as chlorella, unicellular algae Monodus, Eugrena or Diatomaceae have been studied in place of extraction methods from fish oil having drawbacks such as residual fish odor due to incomplete purification and concentration, and the production of EPA using microorganisms has been considered. Recently, fungi producing EPA were reported by Gellerman and Schlenk (J. L. Gellerman and H. Schlenk, BBA, 573, 23, 1979) and Yamada et al. (Meeting of The society of Fermentation Technology, Japan, 1986).

The present inventors sought marine bacteria having an ability to produce EPA to find a new fermentation process for production of EPA using bacteria from which genes can be easily obtained, and which can be cultured in a short time and be easily controlled, and as a result, the present inventors found a new bacteria belonging to the genus Pseudomonas, Alteromonas or Shewanella (K. Yazawa et al., J. Biochem., 103, 5 (1988); K. Yazawa et al., Nippon Suisan Gakkai shi, 54, 1835 (1988)).

It has been suggested that biosynthesis of polyunsaturated fatty acids including EPA works by site-specific aerobic unsaturation of corresponding saturated fatty acids (for example, R. Jeffcoat and A. T. James (1984) in: S. Numa (Ed.): Fatty acid metabolism and its regulation, Elsevier, Amsterdam, pp85 - 112). Moreover, there is a report of WO93/23545-A (EP0594868-A) relating to biosynthetic enzymes which participate in EPA synthesis, and gene coding therefor.

Generally, the ability of a wild strain to produce a useful substance is low, and therefore where it is intended that the ability of the microorganism is industrially used, an improvement, i.e., an increase of productivity of the microorganism is carried out by various methods. The present inventors intended to carry out research for increasing an EPA productivity by finding genes not described in literature for EPA biosynthetic enzymes using gene recombination techniques and introducing the same into another organism, to impart an EPA biosynthesis ability to an organism not having an EPA biosynthesis ability, and eventually to establish an advantageous process for production of EPA.

Accordingly, the present invention provides genes for EPA biosynthetic enzymes, expression plasmids containing said genes, organisms transformed with said plasmid, and a process for production of EPA using said organism.

Figure 1 represents the structure of the plasmid pEPA containing a group of the present genes.

Fig. 2 represents a restriction enzyme map of a DNA fragment containing a group of the present genes.

According to the present invention, an EPA producing strain is constructed by extracting DNA from a microorganism having an ability to produce EPA (microbial origin of genes), cutting this DNA with restriction enzymes to excise genes coded for a group of EPA biosynthesis enzymes, introducing the genes into an appropriate vector to construct an expression plasmid, and transforming a host organism with the plasmid to construct an EPA producing strain. EPA can be produced by this organism.

Although organisms which can be used as a gene source according to the present invention are not limited to specific genera, species or strains, usually, microorganisms classified to the genus Pseudomonas, Alteromonas, Shewanella or the like can be used. These microorganisms can be easily obtained from official or public depository institutes for microorganisms.

As examples of microorganisms belonging to the genus Pseudomonas, Pseudomonas putrefaciens SCRC-2181 (FERM BP-2917), SCRC-2201 (FERM BP-2916), SCRC-2271 (FERM BP-2195), SCRC-2341 (FERM BP-2918), SCRC-2451 (FERM BP-2919), SCRC-2642 (FERM BP-2920), SCRC-2792 (FERM BP-2921), SCRC-2878 (FERM BP-1623), SCRC-3011 (FERM BP-2913), and SCRC-3022 (FERM BP-2914) may be mentioned.

As example of microorganisms belonging to the genus Alteromonas, Alteromonas putrefaciens SCRC-2871 (FERM BP-1624) and Alteromonas putrefaciens subspecies sagamifaciens SCRC-1162 (FERM BP-1626) may be mentioned.

As an example of microorganism belonging to the genus Shewanella, Shewanella putrefaciens SCRC-2874 (FERM BP-1625) may be mentioned.

In the present invention, the case wherein Shewanella putrefaciens SCRC-2874 (FERM BP-1625) was used as a source of genes for a group of EPA biosynthesis enzymes is concretely explained. However, as described above, various EPA producing microorganisms can be similarly used as a gene source. A process for cloning genes is described in the Examples of the present invention.

According to the present invention, EPA producing strains can be artificially generated by transforming an heterogeneous host such as Escherichia coli or a homogeneous host such as Shewanella, or further yeast, fungus or the like. In addition, EPA producing plants can be generated by introducing the present genes into a higher plant such as soybean, sunflower, rape, or the like. As a region for expression of a group of EPA biosynthesis genes, although a control region natively accompanying these enzymes can be used, it is advantageous to prepare another promoter/operator system for increasing an amount of expression or allowing inducible expression. Where E. coli is used as a host, as the promoter/operator system, trp, tac, lavUV5, P_L, P_R or 1pp promoter/operator system and the like can be used, and as an SD sequence, an SD sequence of trp leader peptide, lacZ, metapyrocatechase or cII gene can be used. In addition, a transcriptional terminator, for example, rrnBT₁T₂ terminator of E. coli ribosome gene or the like can be provided downstream of a coding region. In addition, for expression of the above-mentioned gene, a host/vector system of Saccharomyces cerevisiae can be used, wherein as a promoter there can be used a promoter of alcohol dehydrogenase gene, a promoter of acid phosphatase gene, a promoter of glycelaldehyde-3-phosphate dehydrogenase gene, a promoter of enolase gene or the like can be used, wherein a plasmid preferably contains a sequence for replication in yeast, and an auxotrophic marker as a selectable maker for selection of yeast containing said plasmid, such as Leu, Trp, His or the like.

For introduction of the genes into a plant, there are a method using a vector, and a direct introduction method. As vectors, Ti plasmid; DNA viruses such as cauliflower mosaic virus (CaMV), Geminivirus, cassava roten virus, tomato golden mosaic virus, and the like; and RNA viruses such as brome mosaic virus (BMV), and tobacco mosaic virus (TMV) can be used, wherein as a promoter, 35S promoter of CaMV or the like may be mentioned. On the other hand, as direct protoplast introduction methods there can be mentioned a calcium phosphate method, polyethylene glycol method, microinjection, electroporation, liposome method and the like. Moreover, as a direct plant cell introduction method, there may be mentioned a particle gun method.

In addition, a fatty acid composition in a host plant can be changed by using a part of the group of the genes.

Note, that generally, an amount of expression of a particular protein in E. coli is affected by the number of copies of the genes, an efficiency of transcription, stability of mRNA, efficiency of translation, and stability of the protein. To modify control regions such as a promoter, SD region, terminator and the like, a smaller plasmid can be easily treated. The number of copies of the genes depends on the size of the plasmid, and there is a tendency for the plasmid to be smaller as the number of copies increases. For this purpose, a smaller plasmid can be obtained by inserting a DNA fragment containing a group of genes for EPA biosynthesis described in the Examples of the present invention into a plasmid, and repeating subcloning of the plasmid to cut off unnecessary portions of the gene DNA fragment. Smaller EPA biosynthesis enzyme genes thus obtained are included in the present invention. In addition, to enhance stability or activity of the enzyme, the nucleotide sequence of the gene (amino acid sequence) can be modified by a known technique, and the present invention includes such a modified gene.

As a host E. coli of the present invention, any strain derived from E. coli K12 may be used. For example, JM83, JM101, JM103, JM105, JM109, RR1, RB791, W3110, C600, HB101, DH1, AG1, NM554 or the like can be used. As a yeast host, AH22, DC5, D-13-1A, YNN144 or the like can be mentioned.

Note, that a group of the enzymes encoded by the present genes can convert higher fatty acids synthesized by a biosynthetic system natively possessed by the host organism to eicosapentaenoic acid.

In practicing the present invention, an organism such as a microorganism transformed with the present genes is cultured in a medium according to a conventional procedure to obtain microbial cells. In this case, for example, a medium having a composition shown in Table 1 is prepared. Yeast extract 0.5% Pepton 1.0% Sea water 1/2 concentration pH 7.0

From the microbial cells thus obtained, EPA can be obtained according to a conventional procedure such as extraction with an organic solvent. The details are described in the following Examples.

Next, the present invention is explained in more detail by way of Examples.

Shewanella putrefaciens SCRC-2874 (FERM BP-1625) was inoculated in 125 ml of a medium (1% pepton, 0.5% yeast extract, 1/2 concentration artificial sea water), and cultured at 15°C for 18 hours with shaking (OD610 = 8.6). The resulting microbial cells were washed once with 1M NaCl, and suspended in 20 ml of 1M NaCl. The suspension was allowed to stand at 55°C for 30 minutes, 20 ml of 0.1M EDTA was added thereto, and after being allowed to stand at 55°C for 15 minutes, the suspension was centrifuged at 10,000 rpm for 10 minutes. To the precipitate, was added 10 ml of TES buffer (1 mM EDTA, 0.1 mM NaCl, 10 mM Tris-HCl, pH 8.0) containing 100 mg of lysozyme, and the cells were suspended. After being allowed to stand at 37°C for an hour, 1 ml of 10% SDS was added to the suspension, which was then allowed to stand at 60°C for an hour. 11 ml of neutralized phenol was added to the suspension, which was then gently shaken for 5 minutes and centrifuged at 6,500 rpm for 5 minutes to obtain the upper layer. 20 ml of ethanol was added to the layer and the mixture was gently shaken. Precipitated DNA was wound onto a glass bar, washed in ethanol, dissolved in 10 ml of TES buffer, and the solution was allowed to stand at 4°C overnight. 0.5 mg of RNase A was added to the solution, which was then gently shaken at 37°C for 3 hours, and after 1 mg of proteinase K was added thereto, the solution was further shaken for 4.5 hours; 5 ml each of neutralized phenol and chloroform were gradually added to the mixture, which was then gently shaken for 5 minutes and centrifuged to recover the upper layer. 10 ml of chloroform was added to the layer, which was then gently shaken and centrifuged to obtain the upper layer. 20 ml of ethanol was added to the layer, which was then gently shaken, and precipitated DNA was wound onto a glass bar. The DNA was washed in ethanol, and dissolved in 3 ml of TES buffer. An amount of DNA thus obtained was about 2.8 mg. Next, 200 µg of the DNA was partially digested with restriction enzyme Sau3A1 and subjected to electrophoresis on 0.3% agarose, and DNA fragments larger than about 20 Kb were isolated by electroelusion. The DNA fragments were extracted with phenol/chloroform, and precipitated with ethanol, and the precipitate was dissolved in 500 µl of TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4).

As a vector, cosmid pWE15 (STRATAGENE) was used. 10 µg of pWE15 was completely digested with restriction enzyme BamHI, treated with calf intestine alkaline phosphatase at 37°C for an hour, extracted with phenol/chloroform, and precipitated with ethanol, and the precipitate was dissolved in 10 µl of TE buffer. 1.5 µg of the vector DNA thus obtained was mixed with 1 µg of the chromosomal DNA prepared in Example 1 and partially digested with restriction enzyme Sau3A1, and these DNA were ligated using T4 DNA ligase at 26°C for 10 minutes. One fourth of the reaction mixture was packaged according to a conventional method to form phage, which was then infected to E. coli K12/AG-1.

The E. coli suspension infected with the phage of Example 1-2 was plated on an LB agar medium (trypton 1%, yeast extract 0.5%, NaCl 1%, Agar 2%) containing 50 µg/ml ampicillin and cultured at 37°C overnight. The developed colony was inoculated in 1.5 ml of LB medium containing 50 µg/ml ampicillin, and cultured at 25°C for 1 to 7 days with shaking. The culture was centrifuged to collect the microbial cells, and after removing the medium, the cells were suspended in 0.5 ml of methanol saturated with hydrogen chloride. The cell suspension was sealed and incubated at 80°C for an hour to methyl-esterify fatty acids. After allowing the suspension to cool, it was extracted three times with 0.3 ml hexane, the hexane layer was dried and the residue was dissolved in 20 µl of methanol. 2 µl of the solution was spotted on a silica gel plate which was then developed three times with a developing solvent of hexane and ether 19:1, dried in air, and colored with iodine.

In this way, as a result of tests of about 390 recombinant clones, one clone showing a thin layer chromatography spot at the same position as a standard methyl ester of EPA was obtained. From the clone, cosmid was extracted using an alkali/SDS method. This cosmid was designated as pEPA. The pEPA is a cosmid wherein a San3AI fragment of about 38 Kbp was inserted into BamHI site of pWE15.

Cosmid pEPA was prepared from transformant AG-1/pEPA. The pEPA was cleaved with various restriction enzymes, and a restriction enzyme map was prepared (Fig. 1).

An entire nucleotide sequence of a Sau3A1-Sau3A1 fragment containing a genomic DNA insert in the cosmid pEPA is shown in SEQ ID NO: 1. In the nucleotide sequence, 9 open reading frames, ORFs 2 to 10, can be identified, and these nucleotide sequences and corresponding amino acid sequences are shown in SEQ ID NOs: 2 to 10 respectively. The relationship between the entire nucleotide sequence (SEQ ID NO: 1) and ORFs 2 to 10 (SEQ ID NOs: 2 to 10, respectively) is shown in Table 2. SEQ ID NO Length of sequence Positions on SEQ ID NO: 1 2 1983 6121- 8103 4 831 9016- 8186* 6 2910 9681-12590 8 864 13040-13903 10 8268 13906-22173 12 2340 22176-24515 14 6012 24518-30529 16 1629 30730-32358 18 1575 32753-34327 *Reversed sequence extending from the position No. 9016 to the position No. 8186 in the nucleotide sequence shown in SEQ ID NO: 1.

By comparison of the amino acid sequences of various ORFs with known amino acid sequences, it was found that five regions in the ORF 6, and 2 regions in the ORF 8 have homology to some extent with amino acid sequences of enzymes participating in fatty acid synthesis. The results are shown in Table 3. SEQ ID NO (ORF No.) Position of amino acid sequence Similar enzyme and position References 10 (6) 668(Leu)-930(Leu) MalonylCoA-ACP transferase 56(Leu)-309(Leu) (1) 10 (6) 189(Phe)-424(His) Fatty acid synthetase 120(Phe)-350(His) (2) 10 (6) 200(Ser)-483(Leu) Fatty acid synthetase (3-ketoacyl-ACP synthetase domain) 137(Ala)-406(Asp) (3) 10 (6) 204(Ser)-488(Gln) 3-Ketoacyl-ACP synthetase 137(Ala)-406(Asp) (4) 10 (6) 2261(Phe)-2392(Gly) 2-Oxoacylreductase 1470(Leu)-1604(Gly) (5) 14 (8) 205(Ala)-442(Lys) 3-Ketoacyl-ACP synthetase 187(Ala)-416(Asn) (6) 14 (8) 1373(Thr)-1547(Val) 3-Hydroxydecanoyl-ACP dehydratase 29(Leu)-163(Val) (7) References
(1) Magnuson K. et al., FEBS Lett. (1992) 299:262-266
(2) Kameda K. et al., J. Biol. Chem. (1991) 266:419-426
(3) Huang W. Y. et al., Arch. Biochem. Biophys. (1989) 270:92-98
(4) Kauppinen S. et al., Carlsberg Res. Commun. (1988) 53:357-370
(5) Beck J. et al., Eur. J. Biochem. (1990) 192:487-498
(6) Siggaard-Andersen M. et al., Proc. Natl. Acad.Sci. U.S.A. (1991) 88:4114-4118
(7) Cronan Jr. J. E. et al., J. Biol. Chem. (1988) 263:4641-4646

Accordingly, the present invention provides genes coding for amino acid sequences shown in SEQ ID Nos. 4, 8, 10, 14 and 18, as well as DNA coding for amino acid regions having a homology to amino acid sequences of said known enzymes, or coding for amino acid sequences comprising said amino acid regions.

Transformant AG-1/pEPA was inoculated in 100 ml of LB medium containing 50 µl/ml ampicillin, and cultured at 25°C for 48 hours. The cells were obtained by centrifugation, washed once and suspended in 2 ml of pure water, and the suspension was extracted three times with 12 ml of a solvent composed of chloroform and methanol 2:1. The solvent layer was dried, and the residue was dissolved in 1.5 ml of methanol saturated with hydrogen chloride, and the solution was sealed and incubated at 80°C for an hour to methyl-esterify fatty acids. After allowing the solution to cool, it was extracted three times with 2 ml of hexane, and after the hexane layer was dried, the residue was dissolved in 20 µl of methanol. A part of the solution was analyzed by gas chromatography. As a result, a peak of EPA was observed, and a ratio of EPA relating to total fatty acid esters was calculated as about 1.36% from the area of peaks. The amount of EPA per culture volume was about 0.5 mg/l. The ester mixture thus obtained was spotted on an argentation silica gel plate, which was then developed by a solvent composed of hexane and ether at a ratio of 3:1. The plate was colored with fluorescein and ultraviolet light, the spot of the ester of the polyunsaturated fatty acid was scraped off, 1.8 ml of methanol and 0.2 ml of 10% NaCl were added thereto, and the mixture was shaken at room temperature for 30 minutes. The mixture was extracted three times with 2 ml of hexane, the hexane layer was dried, the residue was dissolved in 40 µl of hexane, and GC-MS analysis was carried out. As a result, molecular weight of the substance of the desired peak on the chromatography was 316, and the peak of the fragment conformed to that of an authentic sample, and the substance was identified as EPA. Note that MS fragment peaks were as follow:

Mass:

316(M⁺), 287, 273, 262, 247, 234, 220, 201, 180, 161, 148, 133, 119, 108, 93, 79, 67, 55, 41, 28.

According to a conventional procedure the cosmid pEPA was used to transform E. coli K12/JM109. JM109/pEPA (FERM BP-4257) was obtained by selection using an LB agar medium containing 50 µl/ml ampicillin. According to the same procedure as described in Example 2, extraction of lipid from the cells, methyl-esterification and analysis by gas chromatography were carried out and a peak of EPA was detected. A ratio of EPA relating to a total of esters of fatty acids was calculated as about 1.43% on the basis of the area of peaks. An amount of EPA per culture volume was about 0.6 mg/l.

• Depository Authority: National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology
• Address: 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305 Japan
• Deposition Number and Deposition Date: FERM BP-4257 May 14, 1992

A part of SEQ ID NO: 1 was removed from pEPA by using various restriction enzymes, and the resulting pEPA was rejoined. In addition, an XhoI-SpeI fragment of pEPA was ligated to XhoI-SpeI site of plasmid pBluescript (STRATAGENE) so as to construct pXS-BS. Further, a PacI(9061)-AatII(35564) fragment of pEPA was ligated to PacI-AatII site of plasmid pNEB (New England Biolabs) to construct pPA-NEB. In Addition, an AscI(7710)-AatII(35564) fragment of pEPA was ligated to AscI-AatII site of plasmid pNEB (New England Biolabs) to construct pAA-NEB. These plasmids were used to transform E. coli, and production of eicosapentaenoic acid was tested. Result is shown in Table 4 and Fig. 2. Name of plasmid Position of deletion in SEQ ID NO: 1 Deleted ORF Productivity of EPA pEPAΔ2 XhoI(5666)-AscI(7709) 2 + pEPAΔ4,5 SnaBI(10944)-SnaBI(13226) 4, 5 ** pEPAΔ6 Bbel(16563)-BbeI(20702) 6 - pEPAΔ7 SalI(22265)-NruI(23847) 7 - pEPAΔ8 PstI(24814)-EcoT22I(28946) 8 - pEPAΔ9 SpeI(31446)-SpeI(34626)* 9 - pEPAΔ10 NheI(32526)-SpeI(34630) 10 - pXS-BS Sau3AI(1)-XhoI(5660), SpeI(34632)-Sau3AI(37895) - + pPA-NEB Sau3AI(1)-PacI(9060), AatII(35565)-Sau3AI(37895) 2, 3 - pAA-NEB Sau3AI(1)-AscI(7709) AatII(35565)-Sau3AI(37895) 2 + ** One tenth or less of pEPA * The sequence was inserted in a reverse direction.

From the above-mentioned result, it is suggested that an upstream region of ORF 2, a downstream region of ORF 10, and ORF 2 itself do not participate in the synthesis of eicosapentaenoic acid. Also, it is observed that ORFs 3, 6, 7, 8, 9 and 10 are necessary for the synthesis of eicosapentaenoic acid, and at least one of ORFs 4 and 5 participates in the synthesis of eicosapentaenoic acid.

Among ORFs in the pEPA, ORFs 4, 7, 8 and 9 were separately subcloned into pUC118. For the ORFs 4, 8 and 9, DNA sequence upstream of the translation start codon was shortened by replacing with the synthesized DNA sequence by PCR (polymerase chain reaction). The length of the inserted region and its position for each subclone are shown in Table 5, and primers used for construction of the subclones are shown in Table 6. Plasmid SEQ ID NO Length of Sequence inserted Position in SEQ ID NO: pUCP2 6 3365 9573 - 12937 pUCO5 12 3430 22119 - 25548 pUCP6 14 7083 24364 - 31446 pUCP7 16 2144 30629 - 32772 pUCP8 18 2070 32557 - 34626 Plasmid Primers Sequence of primer (5' → 3') pUCP2 1 AGCTCAAACAACGCGCTTACA 2 TGTTAGTCCCATCACGTTCTTG pUCP6 1 GCCATCATCAGGTGCCATTATCGGT 2 GTCTGGGTAGGCGTGGAAGATT pUCP7 1 AGTATCTGCGTCCTAACTCGAT 2 CCACCTGAATCGGCCTCTG

E. coli JM109/pXS-BS having an ability of synthesize EPA, and JM109 having a subclones constructed in Example 5 were cultured in 50 ml of LB-ampicillin medium at 25°C for 24 hours with shaking. Each culture was centrifuged at 4°C, 3,000 rpm (Hitachi RPR-20-2) for 20 minutes to collect the microbial cells. The cells were suspended in 10 ml of 10 mM PKB (10 mM potassium phosphate buffer, pH 7.0, 2 mM β-mercaptoethanol, 10 mM EDTA), and the suspension was centrifuged at 4°C, 3,000 rpm for 10 minutes to wash the cells. The cell precipitate was suspended in 2 ml of 10 mM PKB, and sonicated. The sonicated cells were centrifuged at 4°C, 33,000 rpm (Beckmann, SW55) for 80 minutes to obtain a supernatant solution, which was then used as an enzyme protein.

A reaction mixture for an enzyme reaction contained [1-¹⁴C]stearoyl-CoA (19 nmoles/µCi) and stearoyl-CoA (total concentration 25 µM), 25 µM malonyl-CoA, 100 µg E. coli acyl carrier-protein (ACP), 1.5 mM NADPH, 1.5 mM NADH, 10 µM cerulenin, 20 µM PMSF and 250 µg of an enzyme protein obtained in Example 6-1, in 0.5 ml of 0.1M potassium phosphate buffer (pH 7.0). A reaction was carried out at 25°C for 30 minutes with shaking.

The reaction mixture was lyophilized overnight, and to the lyophilizate was added 1 ml of 8% HCl-methanol, and the mixture was heated at 80°C for one hour so as to esterify fatty acids produced during the enzyme reaction. The reaction mixture was extracted three times with 1 ml each of n-hexane, and the hexane layer was evaporated in vacuum. The residue was extracted three times with 0.2 ml each of n-hexane. After concentration of the n-hexane extract, a part of the concentrate was co-chromatographed with methyl stearate and methyl arachidate on a reverse phase TLC plate (MERCK RP-8F₂₅₄S), and developed three times with acetonitrile:water (7:1 v/v) for 25 minutes. Distribution of radioisotope on the TLC plate was detected by an AMBIS-RI imaging system and autoradiography.

As a result, the concentrates derived from the enzyme proteins obtained from cultivation of pUCO5 and pUCP6 provided spots near to position of methyl arachidate, which is formed by two carbon-extension of stearic acid.

To identify the spots near to the position of methyl arachidate, radio gas chromatography (RGLC) was carried out using 2m of glass column of 5% synchrome E-71. The detection was carried out with FID (N₂ 60 ml/min.) and an aeration type proportional counter (3,400V) at RI side (CH₄ 250 ml/min.). An amount of about two thirds of the product obtained by at one run of the reaction was applied to the RGLC. As a result, the protein products expressed by the plasmids pUCO5 and pUCP6, which comprise ORF 7 and ORF 8 respectively, provided peak corresponding to methyl arachidate. Radioactivity of the peak is shown in Table 7. Each of these ORFs has an ability to convert stearic acid (C₁₈) to arachidic acid (C₂₀) by 2-carbon extension. Plasmid SEQ ID NO CPM pUCP2 6 15.8 pUCO5 12 42.0 pUCP6 14 29.0 pUCP7 16 9.0 pUCP8 18 9.2